Anti-Tumor Activity of Oleanolic, Ursolic and Glycyrrhetinic Acid Ju-Hong Feng1, Wei Chen1, Yu Zhao2 and Xiu-Lian Ju*,1,3

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48 The Open Natural Products Journal, 2009, 2, 48-52 Open Access Anti-Tumor Activity of Oleanolic, Ursolic and Glycyrrhetinic Acid Ju-Hong Feng1, Wei Chen1, Yu Zhao2 and Xiu-Lian Ju*,1,3

1Hubei Key Laboratory of Novel Reactor & Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China 2Department of Traditional Chinese Medicine and Natural Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, China 3Key Laboratory for Green Process of Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, China

Abstract: Over last few years, there has been great interest on the anti-tumor properties of the triterpenoids, oleanolic, ursolic and glycyrrhetinic acid in presence of many plants such as Olea europeae L., Actostaphylos uva-ursi and Glycyr- rhiza uralensis. The three acids and their derivatives show potential anti-tumor promoting and cytotoxic activities, inhibiting proliferation, inducing apotosis and preventing invasion, which suggested that they could be developed as anti-cancer and cancer chemopreventive agents. The mechanisms of the anti-tumor effects by triterpenoids need further investigation. A brief review is attempted here for their anti-tumor activities and chemical modification research. Keywords: Oleanolic, ursolic, glycyrrhetinic acid, derivatives, anti-tumor.

INTRODUCTION published, reflecting the huge interest and the progress made in the understanding of these substances, including the isola- Oleanane and ursane triterpenoids are pentacyclic com- tion and purification of triterpenoids from various plants, pounds with 30 carbon atoms, biosynthetically derived from chemical modifications, pharmacological studies on their the cyclization of squalene [1]. These natural products, beneficial effects, toxicological studies, and the clinical use whose structural diversity includes a wide array of functional groups, abound in rosemary, thyme, oregano and lavender, for the treatment of different diseases, e.g., anticancer che- e.g. Olea europeae L., Swertia mileensis T., Ligustrum lu- motherapy [4]. cidum Ait. and Actostaphylos uva-ursi [2]. ANTI-TUMOR PROMOTING ACTIVITY Oleanolic acid (3-hydroxy-olean-12-en-28-oic acid, OA, 18-GA, UA and their 3-keto derivatives were stud- OA) (Fig. 1), an oleanane triterpenoid and ursolic acid (3- ied by Ohigashi’s group for their effects on 12-O- hydroxy-urs-12-en-28-oic acid, UA) a ursane triterpenoid, tetradecanoyl-phorbol-13-acetate (TPA)-induced Epstein- are ubiquitous triterpenoids in plant kingdom, medicinal Barr virus (EBV) activation in Raji cells [5]. Both OA and herbs, and are integral part of the human diet [3]. OA and UA significantly inhibited the activation and the dose re- UA have the similar chemical structures but differ only in sponses of the acids were very similar to those of the known the position of the methyl group in ring E. OA has two anti-tumor promoter, retinoic acid. Furthermore, enhance- methyl groups at its C-20 position while UA has a respective ment of the inhibitory activity was found in 3-keto deriva- methyl group at C-19 and C-20 positions. tives of OA and UA while oxidation at C-3 of GA led to 18-Glycyrrhetinic acid (3-hydroxy-11-oxoolean-12-en- reduction of the activity. Huang’s team also reported that the 28-oic acid, GA) (Fig. 1), another oleanane-type triterpenoid, 3-keto derivatives of OA might be a useful anticancer agent is very structurally similar with oleanolic acid except that it for melanoma [6]. has carbonyl group at C-11 and its carboxyl moiety at C-20 In 1986, OA and UA were tested against inhibitory effect positions. It and its regioisomer –18-glycyrrhetinic acid on tumor promotion by TPA in vivo [7]. They inhibited ef- both exist in Glycyrrhiza uralensis in the form of free acid or fectively the tumor promotion in mouse skin and the role of saponin. The saponin, called glycyrrhizic or glycyrrhizin UA for inhibitory action on tumor promotion might differ (GL) is composed of glycyrrhetinic acid and two moleculars slightly from those of OA and retinoic acid. Using the same of glucuronate. bioassay technique, the following reports suggested that These natural triterpenoids mentioned above are structur- some oleanone and ursane diols and triols could be useful as ally closely and share many common pharmacological prop- chemopreventive agents [8, 9]. erties. Over the last decade, hundreds of articles have been It’s reported that 18-GA was more effective than 18- GA to inhibit the mutagenicity of benzo[a]pyrene, 2-

*Address correspondence to this author at the Hubei Key Laboratory of aminofluorene and aflatoxin B1 in Salmonella typhimurium Novel Reactor & Green Chemical Technology, School of Chemical Engi- TA98 and TA100 [10]. The acids exhibited substantial anti- neering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, skin tumor initiating and anti-skin tumor promoting activi- China; Tel: +86-27-87194980; Fax: +86-27-87194465; ties. E-mail: [email protected]

1874-8481/09 2009 Bentham Open Anti-Tumor Activity of Oleanolic, Ursolic The Open Natural Products Journal, 2009, Volume 2 49

30 29 19 21 20 E 12 18 11 13 17 22 25 26 C COOH COOH 1 DH H 14 28 9 2 16 A B 15 10 8 3 27 4 HO 5 7 HO 6 23 24 ursolic acid oleanolic acid COOH COOH

O O H H

HO HO

18-glycyrrhetinic acid 18-glycyrrhetinic acid Fig. (1). Structures of the triterpenoids.

CYTOTOXIC ACTIVITY system postirradiation in mice [16]. UA was more potent tumorigenic inhibitor than OA. The cytotoxic activities of these triterpenoids and their derivatives were investigated widely. OA and UA as the anticancer constituents were isolated from Pterocarya tonkinesis by a bioassay using tsFT210 UA and its related derivatives were tested for their cyto- cells [17]. The acids inhibited the proliferation of human toxic activity on lymphocytic leukemia cells P-388, L-1210, erythroleukemia K562 cells with the inhibition rates of human oral epidermoid carcinoma KB, human lung carci- -1 42.4% and 33.7% at 10 mg·L , respectively. noma cell A-549, human ileocecal carcinoma HCT-8 and human mammary gland carcinoma MCF-7 tumor cell lines The anti-tumor activities of OA, 18-GA and UA were [11]. Esterification of the hydroxyl group at C-3 and the car- studied by Huang’s group in detail. The research on the pro- boxyl group at C-17 positions led to compounds with de- liferation inhibition and differentiation induction of 18-GA creased cytotoxicity in the human tumor cell lines, but with and GL on human hepatocarcinoma cells BEL-7402 has equivalent or slightly increased activity against the growth of been reported [18]. In the same effect, the dosage of 18-GA L-1210 and P-388 leukenic cells. is lower about 40 times than that of GL. Subsequently the The cytotoxicity activities of OA and UA on Jurkat cell inhibitory effect of GL, 18-GA, OA and UA on the prolif- line (T cell lymphoma) were reported [12]. The results sug- eration and invasion of the human lung cancer cells PGCL3 gested that UA and OA have significant anti-tumor activity were investigated [19]. The mechanism of anti-invasion might be to inhibit the adhesion, migration and the CB secre- and UA is more effective than OA with IC50 values of 75 μM and 150 μM, respectively. The anti-tumor mechanism of the tion of the cells. Both OA and UA could decrease the prolif- acids was supposed to be killing the cells by cytotoxin with eration of PGCL3 cells, and their IC50 values were 40.71 μM high dose (100 μM) and inhibiting the proliferation of cells and 44.73 μM respectively. The combination of retinoic acid with low dose (50 μM). and 18-GA had synergistic effect against the proliferation of PGCL3 cells [20]. UA, 3-keto derivatives of OA and UA isolated from the aerial roots of Ficus microcarpa were tested their cytotoxic The mechanism of UA induced effects on K562 cells was activities in vivo using three human cancer cell lines, namely, reported [21]. The results showed that UA inhibited K562 HONE-1 nasopharyngeal carcinoma, KB oral epidermoid cells proliferation in a dose-dependent manner. Apparent carcinoma, and HT29 colorectal carcinoma cells [13]. These morphological index of apoptosis was observed. Expression three natural products showed remarkable cytotoxicity of Bcl-2 was down-regulated, but Bax expression was en- against the cell lines. hanced. Activation of Caspase-3 and down-regulation of phosphotyrosine was determined. These results indicated The effects of UA on the proliferation of mouse melanoma cell line B16 were studied by Es-saady group and it that UA could induce apoptosis in K562 cells in vitro. Up- regulation of Bax and activation of Caspase-3 might contrib- inhibited B16 proliferation with an IC50 value of 10 μM by MTT technique [14]. The derivatives of OA and UA could ute to this effect. also inhibit the proliferation of cell lines such as WI-38 lung CHEMICAL MODIFICATION fibroblast cells, VA-13 malignant tumor cells and HepG2 human liver tumor cells [15]. Efforts have been made to produce more effective and less toxicity derivatives of these triterpenoids by chemical OA and UA were examined for their ability to inhibit the modification (Fig. 2). tumor growth and enhance the recovery of hematopoietic 50 The Open Natural Products Journal, 2009, Volume 2 Feng et al.

R1 H H COOCH3

ref. [22] 1 R = H2, O R1 ref. [24] R2= COOCH , CH OH 3 2 R1= -H -OH, O O COOCH3 1 CH2O P R

N(CH CH Cl) R2 2 2 2 H H O ref. [23] O R1 P O 1 R1 P R = OH, NH2 O ref. [23] 2 N(CH2CH2Cl)2 R = O, H2 1 N(CH2CH2Cl)2 R = OH, NH2

O OCH3

ref. [25] O R2 R1= -H -OH, -H -OAc, 3 -H -NH , O, N-OH H R 2 O 2 R = O, H2 3 H R = COOCH3, COOH, O CONH (CH ) NH ref. [26] R1 2 2 9 2 HO GA-DZ

O O

R2 R1 H COOH NC H

ref. [31] O O 1 R = CN, COOCH3, COOH, ref. [32] CHO, CONH2, H CDDO 2 R = COOH, COOCH3 Fig. (2). Strucures of OA, UA and GA derivatives by chemical modification.

Terasawa’s group reported a series of modified deriva- anti-tumor activities in comparison with the nitrogen mus- tives of 18- and 18-GA and their anti-tumor-promoter tard. activities of TPA in vitro [22]. The structure-activity rela- Oxidation of UA on its 1, 2 and 3- positions was carried tionships (SAR) study showed that the predominant factors out and the models of Hela human cervical carcinoma cells for the high anti-tumor-promoter activity in GA series com- and HL-60 cells were used for bioactive screening [24]. The pounds to be the hydrophobic character of the ring A moiety, results indicated that introduction of the oxidized substituent the presence of an 11-oxo function, and the introduction of a groups in ring A of UA led to the decrease of anti-tumor 20-hydroxymethyl group. No essential difference was found activities. for inhibitory effects between the 18- and 18-series. Structural modifications were performed on the C-3, C- Six new GA derivatives were synthesized from the reac- 28 and C-11 positions of UA and the cytotoxicity of the de- tion of di(2-chloroethyl)-aminophosphonyl dichloride with rivatives was evaluated [25]. The SAR study revealed that methyl glycyrrhetinate, methyl-11-deoxy-glycyrrhetinate, the triterpenoids possessing two hydrogen-bond forming and olean-12-en-3,30-diol, respectively [23]. The prelimi- groups (an H-donor and a carbonyl group) at C-3 and C-28 nary anti-tumor activity against EAC cells both in vitro and positions exhibit cytotoxic activity. A 3-amino derivative in vivo showed that the new compounds exhibited potent was 20 times more potent than the parent UA. Anti-Tumor Activity of Oleanolic, Ursolic The Open Natural Products Journal, 2009, Volume 2 51

1 R R1 R2 R2 R3 HO H H

HO 1 R = OCOCH3, OH 2 R = H2, O 1 R = H , O 3 2 R = COOH, COOCH3 2 R = COOH, COOCH3

1 1 H R R

HO HO 1 R =COOH, COOCH3, CH2OH Fig. (3). Structures of OA derivatives synthesized by the authors.

In an in vitro anti-cancer assay, using nine different hu- entiation. Finally, OA, UA and GA are capable of interfer- man tumor cell lines, the GA-DZ (dehydrozingerone) conju- ence with many processes going on in normal and malignant gates showed significant cytotoxic effects [26]. Similar con- cells. However, many aspects of their biological activities jugates between DZ and OA or UA were inactive suggesting are not understood properly. The mechanisms of the anti- that the GA component is critical for activity. tumor effects by triterpenoids need further investigation. Since 1997, Honda’s group had commenced the research Our research group has been interested in the SAR study on the inhibitory effect of UA and OA on nitric oxide (NO) of OA, UA and GA. We had designed and synthesized more production induced by interferon- (IFN-) in mouse macro- than one hundred derivatives of these triterpenoids, which phage [27-31]. Excessive production of NO can destroy have dihydroxyl groups with different configurations, enone functional normal tissues during acute and chronic inflam- moieties, allylic alcohol functionalities, along with conju- mation. This phenomenon is also closely related mechanisti- gated homoannular and heteroannular diene frames (the cally to carcinogenesis. Thus, inhibitors of NO production in structures of OA derivatives illustrated in Fig. 3). The pre- macrophage are potential anti-inflammatory and cancer liminary biological assays showed that some derivatives had chemopreventive drugs. About sixty OA and UA derivatives more potent cytotoxic activity than cisplatin against KB, were initially randomly synthesized to test their inhibitory of BEL7404, A549, HL-60, CNE and PC-3 cell lines [33]. The NO production in mouse macrophage. The results provided further SAR research has been in process. the following interesting SAR: 1) in the A ring, a 1-en-3-one functionality was important for significant activity; 2) the REFERENCES oleanane skeleton was more potent than the ursane skeleton; [1] Connolly, J.D.; Overton, K.H. Chemistry of Terpenes and Terpe- 3) carboxyl, methoxycarbonyl and nitrile groups at C-2 en- noids. Newman Academic Press: New York, 1972. hanced activity, however, hydroxyl, aminocarbonyl, [2] Ovesná, Z.; Vachálková, A.; Horváthová, K.; Tóthová, D. Neo- plasma, 2004, 51, 327-333. methoxy, chloride, and bromide groups decreased activity [3] Somova, L.O.; Nadar, A.; Rammanan, P.; Shode, F.O. Phytomedi- while a formyl group did not confer activity but only toxic- cine, 2003, 10, 115-121. ity; 4) a 9(11)-en-12-one functionality was the strongest en- [4] Liu, J. J. Ethnopharmacol., 2005, 100, 92-94. hancer of potency among structures of ring C. The selected [5] Ohigashi, H.; Takamura, H.; Koshimizu, K.; Tokuda, H.; Ito, Y. Cancer Lett., 1986, 30, 143-151. oleanane triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)- [6] Tokuda, H.; Ohigashi, H.; Koshimizu, K.; Ito, Y. Cancer Lett., dien-28-oic acid (CDDO) was found to be a potent, multi- 1986, 33, 279-285. functional agents in various in vitro assays [32]. [7] Huang, D.; Ding, Y.; Lia, Y.; Zhang, W.M.; Fang, W.S.; Chen, X.G. Cancer Lett., 2006, 233, 289-296. CONCLUSION [8] Nishino, H.; Nishino, A.; Takayasu, J.; Hasegawa, T.; Iwashima, A.; Hirabayashi K.; Iwata, S.; Shibata, S. Cancer Res., 1988, 48, The triterpenoids, OA, UA and GA have attracted con- 5210-5215. siderable interest because of their low cytotoxicity and their [9] Ukiya, M.; Akihisa, T.; Tokuda, H.; Suzuki, H.; Mukainaka, T.; range of biological activities [4]. Over the last few years, Ichiishi, E.; Yasukawa, K.; Kasahara, Y.; Nishino, H. Cancer Lett., there has been great interest on the part of researchers in the 2002, 177, 7-12. [10] Wang, Z.Y.; Agarwal, R.; Zhou, Z.C.; Bickers, D.R.; Mukhtar, H. chemopreventive, cytotoxic properties of the three triterpe- Carcinogenesis, 1991, 12, 187-192. noids. According to Ovesna et al. [2], these triterpenoids and [11] Lee, K.H.; Lin, Y.M.; Wu, T.S.; Zhang, D.C.; Yamagishi, T.; Ha- their derivatives can act on various stages of tumor devel- yashi, T.; Hall, H.I.; Chang, J.J.; Wu, R.Y.; Yang, T.H. Planta opment, including the inhibition of tumorigenesis, inhibition Med., 1988, 54, 308-311. during tumor promotion, and induction of tumor cell differ- 52 The Open Natural Products Journal, 2009, Volume 2 Feng et al.

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Received: October 20, 2008 Revised: November 08, 2008 Accepted: February 24, 2009

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